The biosynthesis of isopentenyl diphosphate (IPP) is essential for the production of a broad variety of isoprenoids that serve crucial roles in membrane stability, defense and communication, photoprotection, and sugar transport. Recently, a novel branch of the mevalonate pathway was discovered in the archaeon Methanocaldococcus jannaschii involving an enzyme called isopentenyl phosphate kinase (IPK) that could phosphorylate isopentenyl monophosphate to isopentenyl diphosphate.
Isopentenyl diphosphate (IPP) is the central precursor to a diverse collection of isoprenoids and isoprenoid-derived compounds present in many different organisms. Following its biosynthesis, successive units of IPP are used with either dimethylallyl diphosphate (DMAPP) or a growing isoprenoid diphosphate to synthesize C10, C15, or C20 oligoprenyl diphosphates known as geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP), respectively. These three isoprenoid diphosphates are the building blocks for many downstream biosynthetic compounds that serve a colorful variety of roles amongst the different kingdoms of life. All three of them can be cyclized by their respective terpene cyclase to generate an astounding selection of volatile terpenes which are extremely important for defense and communication in plants, fungi, several insects, certain bacteria, and marine organisms (Gershenzon & Dudareva, 2007, Nat. Chem. Biol. 3:408-414.). FPP is the most ubiquitous of the three building blocks and is transformed into a variety of essential biomolecules throughout all kingdoms of life. Some of these biomolecules include squalene, hopanoids, and steroids (which are important for membrane structure in Archaea, Bacteria, and Eukarya, respectively) (Novakova et al., 2008, Folia Microbiol. (Praha) 53:237-240; Ourisson et al., 1987, Annu. Rev. Microbiol. 41:301-333), and dolichols, which serve a critical role in N-glycosylation and membrane anchorage of sugars in eukaryotes and archaea (Eichler & Adams, 2005, Microbiol. Mol. Biol. Rev. 69:393-425). GGPP is the precursor to all carotenoids, which are important for photoprotection in many plants, fungi, algae, bacteria and some archaea (Sieiro et al., 2003, Int. Microbiol. 6:11-16; Hemmi et al., 2003, Biochem. Biophys. Res. Commun. 305:586-591). Interestingly, GGPP is also a precursor to the isoprenoid-derived hydrocarbon moiety of lipids that are present exclusively in Archaea. See Koga & Morii, 2007, Microbiol. Mol. Biol. Rev. 71:97-120 for a review on archaeal lipids.
It is clear that IPP is a necessary building block for all downstream isoprenoids and it is essential for the survival of any organism. It is therefore crucial that we understand how this molecule is produced in various organisms. There are two known pathways that ultimately produce IPP and DMAPP (the other precursor to all downstream isoprenoid products): the mevalonate (MVA) pathway and the more recently discovered 1-deoxy-d-xylulose-5-phosphate (DXP) pathway (Rohmer, 1999, Nat. Prod. Rep. 16:565-574). The MVA pathway is utilized by animals, plants (cytosol), fungi, and certain bacteria, while the DXP pathway is found within plants (plastids), cyanobacteria, and certain parasitic organisms (Lange et al., 2000, Proc. Natl. Acad. Sci. U.S.A. 97:13172-13177). In archaea, homologs for many of the genes in the MVA pathway have been found; however, the two last genes leading up to IPP biosynthesis (normally encoding phosphomevalonate kinase and diphosphomevalonate decarboxylase) are missing. See FIG. 6. For this reason, the isoprenoid pathway in archaea has been referred to as “The Lost Pathway” (Smit & Mushegian, 2000, Genome Res. 10:1468-1484). Attempts to reconstruct The Lost Pathway have only recently shown promise. In 2006, a group discovered an enzyme present in the archaeon Methanocaldococcus jannaschii that was able to phosphorylate isopentenyl monophosphate, thereby producing IPP (Grochowski, et al., 2006, J. Bacteriol. 188:3192-3198). This protein, named isopentenyl phosphate kinase (IPK), not only allows for the partial reconstruction of The Lost Pathway, but also represents a completely unique branch of the universal mevalonate pathway. This is a fascinating discovery considering the fact that archaea, when compared with the other two domains from which life originated, have evolved distinct functions for isoprenoid compounds.
Isopentenyl phosphate kinase shares significant sequence homology with the amino acid kinase (AAK) superfamily. See FIG. 7. Members of this family usually utilize magnesium and ATP to phosphorylate small molecule substrates that contain carboxylate, carbamate, phosphonate, or phosphate functional groups. Disclosed herein, inter alia, is the crystal structure of isopentenyl phosphate kinase from M. jannaschii solved in its apo form and in complex with substrate. These structures coupled with the biochemical analysis of several mutants suggest an important role for an active site histidine residue which is not conserved among all AAK family members and has not previously been assigned a role in catalysis.